| Tobacco(Nicotiana tabacum L.)is one kind of important cash crops in China.However,tobacco brown spot,caused by Alternaria alternate(Fries)Keisslf.sp.nicotianae,is a more destructive foliar disease in tobacco production areas all over the world,which induces serious effect on yield and quality of tobacco leaves.Given its unique agricultural impacts,it is an urgent need to study the resistance mechanism,so as to select disease-resistant cultivars and control disease for tobacco production.In this study,the resistant tobacco cultivar ‘JYH',a susceptible one ‘CBH' and their hybrids were selected as experimental materials to systematically investigate the genetic mechanism.In the meanwhile,the physiological and biochemical characteristics between resistant and susceptible cultivars under tobacco brown spot stress were also analyzed,which including the endogenous hormones changes,the differences in carbon and nitrogen metabolism and phenolic metabolism,and the responses of photosynthesis,PSII photochemical characteristics,in addition,the transcriptome sequencing techniques were also used to detect the expression changes of transcription factors and their families in two cultivars,so as to provide basic theory for the disease resistance mechanism between host and pathogen.The main results were showed as following:1.Inheritance analysis of resistance to tobacco brown spotIn order to further understand the genetic mechanism of resistance to tobacco brown spot,six populations(P1,F1,P2,BC1,BC2 and F2)derived from the same cross of ‘CBH?JYH' were applied to investigate the inheritance law by means of the mixed major gene and polygene inheritance model with joint analysis method of multiple generations.The results showed the resistance inheritance of JYH to tobacco brown spot was a quantitative trait and it fitted the mixed genetic model of two pairs of additive-dominant-epitasis major genes plus additive-dominant-epitasis polygene(the E-0 model).In addition,the first pair gene showed negative partial dominance,and the second was negative super-dominance,at the same time,the additive,and epistatic effects of two major genes all played an important role.In the populations of BC1,BC2 and F2,the major genes heritability were estimated to be 78.36%,69.78% and 92.36%,respectively,and the polygene heritability to be 13.53%,20.22% and 1.73%,while the ratios of the environmental variance to phenotype variance were 5.91-9.99%.In conclusion,the tobacco brown spot resistance was controlled by two pairs of additive-dominant-epitasis major genes plus additive-dominant-epitasis polygene,and also affected by environmental variation.Therefore,major gene resistance ought to be paid more attention,and polygenetic effects should be also considered for the breeding of resistant cultivars.2.Changes of endogenous hormones in two tobacco cultivars with different resistance caused by tobacco brown spot stress The effects of tobacco brown spot stress on endogenous hormones were studied at pot experiment condition,with the purpose to find their relations to disease resistance.The results showed that,the endogenous hormones contents varied under tobacco brown spot stress,and there was differences existed between the resistant and susceptible cultivars.Compared with the control,the contents of ABA and ethylene production in two cultivars all had an upward trend under stress,whereas the IAA and CTK contents declined sharply.In the meanwhile,tobacco brown spot stress increased the contents of GA and JA in JYH,on the contrary,the GA content in CBH had change trend of fall after the rise at first,and the JA content continued to decline during the stress period.Under tobacco brown spot stress,the smaller variations of endogenous hormones contents in resistant cultivar was useful to improve its disease resistance,so as to keep the normal growth.The endogenous hormone metabolism in susceptible cultivar was disorder influenced by tobacco brown spot,with the result of a susceptibilty.These results indicated there was a close relationship between endogenous hormones and disease resistance.It is important for plants to keep the balance of endogenous hormones to reduce the harm caused by tobacco brown spot.3.Effects of tobacco brown spot stress on carbon and nitrogen metabolism in two tobacco cultivars with different resistanceThe responses of carbon and nitrogen metabolism were investigated under tobacco brown spot stress at pot experiment condition,to discuss the influence mechanism on tobacco growth caused by tobacco brown spot.The results showed as following,tobacco brown spot stress decreased the activities of GOGAT and SPS in two cultivars,whereas the ammonium nitrogen,total nitrogen and starch were accumulated under stress,in addition,the SS activity and the contents of nitrate nitrogen,sucrose and soluble sugar increased firstly and then declined.Under tobacco brown spot stress,CBH had a sustained downward trend in the activities of NR,GS,GDH,AM and INV,in contrast,those activities in JYH rose at the beginning and then dropped with the increasing stress degree,and the GS activities decreased firstly and increased subsequently.Those results suggested the higher ability of carbon and nitrogen metabolism had positive correlation with the increased disease resistance to tobacco brown spot.Under stress,the resistant cultivar had higher activities of key enzymes in carbon and nitrogen metabolism,the ammonium nitrogen content was lower,and the carbohydrate metabolism was active,which helped to strengthen its resistance to tobacco brown spot,whereas,in susceptible cultivar,the activities of key enzymes were seriously influenced,the contents of ammonium nitrogen and total nitrogen were excessively accumulated,and the ability of carbohydrate utilization also reduced,which led to the disorder of carbon and nitrogen metabolism under tobacco brown spot stress,in the consequence of the aging of tobacco plant death.cultivars with different resistance caused by tobacco brown spot stress.4.Responses of photosynthesis,PSII photochemical characteristics in two tobaccoThe changes of photosynthetic pigment contents,photosynthetic and PSII photochemical characteristics in resistant and susceptible cultivars were investigated under three different tobacco brown spot stress levels,with the purpose to find features on the utilization and conversion of light energy in tobacco cultivars with different resistance under tobacco brown spot stress.The results showed was that,tobacco brown spot stress could decrease the total chlorophyll,chlorophyll a,chlorophyll b and carotenoids in two cultivars,in addition,the decreasing range in CBH was bigger than that in JYH.Tobacco brown spot also reduced net photosynthetic rate(Pn)and stomatal conductance(Gs)in JYH and CBH,however,the Pn in JYH increased under mild stress.The changes of intercellular CO2 concentration(Ci)and stomatal limitation value(Ls)in two cultivars were obviously different,the Ci in CBH tended to elevate under tobacco brown spot stress,while the Ls had a significantly decline,and the two parameters in JYH under severe stress had the same variation trend as those in CBH.Compared with the rising trend of Ls,the Ci in JYH decreased under mild and moderate stress.With the upward levels of tobacco brown stress,the initial fluorescence(F0)and non-photochemical quenching(NPQ)in two cultivars both increased,in contrast,the F0 in two cultivaes under severe stress showed an obvious increase.However,the maximal fluorescence(Fm),variable fluorescence(Fv),maximal photochemical efficiency(Fv/Fm),PSII potential efficiency(Fv/F0),photochemical quenching coefficient(qp)and PSII actual quantum yield(?PSII)in two cultivars all decreased under tobacco brown spot stress,and the decreased range was bigger in CBH than that in JYH.In addition,JYH had a higher PSII electron transport rate under stress.These results indicate that,the photosynthetic pigments,photosynthesis and PSII photochemical activities in resistant cultivar were less influenced than those in susceptible one under tobacco brown spot stress,so as to have higher photosynthetic performance,which were the main physiological features in the adapt ability to tobacco brown spot stress.5.Changes on phenolic metabolism in two cultivars with different resistance under tobacco brown spot stressThe activities of phenylalanine ammonialyase(PAL),peroxidase(POD)and polyphenol oxidas(PPO),and the contents of chlorogenic acid,flavonoids,total phenols and lignin were investigated under tobacco brown spot stress,in order to elucidate the relationship between phenolic metabolism and disease resistance.The results showed as following,under tobacco brown spot stress,the activities of PAL and PPO and the contents of chlorogenic acid,flavonoids,total phenols and lignin in two cultivars with different resistance all had the same variation trend,which increased firstly and then declined gradually with the rising stress degree,on the contrary,the POD activity rose sharply.JYH had higher activities of PAL,POD and PPO than CBH,and the contents of chlorogenic acid,flavonoids,total phenols and lignin were also bigger.The relevant analysis results showed,under stress of JYH,a significant positive correlation was found between the PAL activity and lignin content,the POD activity and chlorogenic acid content.In the meanwhile,the PAL activity in CBH was positively related to total phenols content,and the correlation between PAL activity and the contents of chlorogenic acid,flavonoids and lignin all had a bigger increase.Under tobacco brown spot stress,the phenolic metabolism has an important effect in its resistance to tobacco brown spot.In resistant cultivar,the key enzyme activities in related with phenolic metabolism all increased significantly,and had higher contents of phenolic compounds,which played important roles in enhancing the resistance to tobacco brown spot.6.Transcription analysis in two cultivars with different resistance under tobacco brown spot stress The expression changes of transcription factors and their families in two tobacco cultivars were studied under tobacco brown spot stress,so as to definite the regulatory mechanism of transcription factors in response to tobacco brown spot stress.The results showed that 59 differential expressed transcription factors were investigated in resistant cultivar under tobacco brown spot stress,in addition,its up-regulated transcription factors was 23,and the number of down-regulated transcription factors reached 36.In susceptible cultivar,the expression of 46 transcription factors had changed,and the number of up-regulated transcription factors and down-regulated transcription factors was 46 and 18,in separately.There were 31 overlapping transcription factors exhitited the same trend in two cultivars under tobacco brown spot stress,with 28 transcription factors in resistant cultivar and 15 factors in susceptible cultivar were cultivar-special in response to stress respectively.The transcription factor families including WRKY,MYB,AP2/EREBP,b ZIP,Homeodomain and b HLP in two cultivars under tobacco brown spot stress had more transcription factors than othe families,the up-regulated transcription factor families were WRKY,MYB,AP2/EREBP and b ZIP in both resistant and susceptible cultivar,whereas,the families of MYB,AP2/EREBP,b ZIP,Homeodomain and b HLP belonged to down-regulated transcription factor families.At the same time,compared with susceptible cultivar,the three families of NAC,HSF and PHD were unique to resistant cultivar,and they all had a down-regulated trend.In conclusion,the number of up-regulated together with down-regulated transcription factors in resistant cultivar is higher than that in susceptible one under tobacco brown spot stress,and the disease resistance difference among tobacco cultivars was related to the expression changes of transcription factors. |
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